Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis
Site directed mutagenesis is a molecular biology technique used to determine the biological roles of many proteins. It complements computational techniques such as protein docking, where the structure of a complex between protein and its substrate is predicted based on the independently crystallized...
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sg-ntu-dr.10356-164832023-03-03T15:32:39Z Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis Choong, Carmen. Jiang Rongrong School of Chemical and Biomedical Engineering DRNTU::Engineering::Chemical engineering::Biotechnology Site directed mutagenesis is a molecular biology technique used to determine the biological roles of many proteins. It complements computational techniques such as protein docking, where the structure of a complex between protein and its substrate is predicted based on the independently crystallized structures of the components through X-ray crystallization. Site directed mutagenesis involves a desired mutation at a defined site in a DNA molecule, and it is the molecular technique investigated in this research. The substrate specificity of hydrogen peroxide forming NADH oxidase from Salmonella typhimurium (S. typhimurium) was changed based on the protein docking results by doing site directed mutation. Two potential amino acids, Glutamic acid at position 385 and Phenylalanine at position 386, which were likely to be responsible for binding to NADH for oxidation to form hydrogen peroxide, were mutated to Serine and Arginine respectively through site directed mutagenesis. The gene, alkyl hydroperoxide reductase subunit F (AhpF), has been cloned into two types of vectors: pJET1 for storage of gene, and pET30b(+) for protein expression. Activity of the mutated gene will be measured to determine the oxidation of AhpF in the presence of NADPH as a substrate. Bachelor of Engineering (Chemical and Biomolecular Engineering) 2009-05-26T07:48:40Z 2009-05-26T07:48:40Z 2009 2009 Final Year Project (FYP) http://hdl.handle.net/10356/16483 en Nanyang Technological University 69 p. application/pdf |
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DRNTU::Engineering::Chemical engineering::Biotechnology Choong, Carmen. Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis |
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Site directed mutagenesis is a molecular biology technique used to determine the biological roles of many proteins. It complements computational techniques such as protein docking, where the structure of a complex between protein and its substrate is predicted based on the independently crystallized structures of the components through X-ray crystallization. Site directed mutagenesis involves a desired mutation at a defined site in a DNA molecule, and it is the molecular technique investigated in this research.
The substrate specificity of hydrogen peroxide forming NADH oxidase from Salmonella typhimurium (S. typhimurium) was changed based on the protein docking results by doing site directed mutation. Two potential amino acids, Glutamic acid at position 385 and Phenylalanine at position 386, which were likely to be responsible for binding to NADH for oxidation to form hydrogen peroxide, were mutated to Serine and Arginine respectively through site directed mutagenesis. The gene, alkyl hydroperoxide reductase subunit F (AhpF), has been cloned into two types of vectors: pJET1 for storage of gene, and pET30b(+) for protein expression. Activity of the mutated gene will be measured to determine the oxidation of AhpF in the presence of NADPH as a substrate. |
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Jiang Rongrong |
author_facet |
Jiang Rongrong Choong, Carmen. |
format |
Final Year Project |
author |
Choong, Carmen. |
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Choong, Carmen. |
title |
Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis |
title_short |
Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis |
title_full |
Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis |
title_fullStr |
Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis |
title_full_unstemmed |
Change hydrogen-peroxide forming NADH oxidase substrate specificity by site directed mutagenesis |
title_sort |
change hydrogen-peroxide forming nadh oxidase substrate specificity by site directed mutagenesis |
publishDate |
2009 |
url |
http://hdl.handle.net/10356/16483 |
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1759853503586500608 |